Project summary: Notchl and Notch2 are redundantly required during mammalian development and stem cell differentiation in different tissues, yet each protein has acquired the ability to perform unique functions that cannot be provided by other co-expressed Notch proteins. This implies a layer of regulation unique to vertebrates whose molecular mechanism is unknown. We propose to elucidate the basis for the differential Notchl and Notch2 functions by identifying endogenous target genes. To facilitate this experiment we plan to knock-in epitope C-terminal tags unique for each Notch. Chromatin immunoprecipitation of direct physiological Notch target genes from Wt cells coupled with microarray analysis of gene expression in Notchl- or Notch2-deficient cells will identify differentially regulated targets and allow us to determine whether Notch proteins carryout distinct functions through binding to unique targets or by differential activation of shared targets. In silico analysis and proteomic approaches will be deployed to identify potential collaborating factors specific for each Notch protein. Furthermore, the analysis of the Notchl and Notch2 deficient ES cells will be informative of their respective roles in the differentiation process. Future studies will entail generating mice from the epitope tagged Notch knock-in ES cells to confirm endogenous target genes in vivo through similar chromatin immunoprecipitation experiments in tissues that display distinct functions of Notchl and Notch2. Additionally, microarray analysis of gene expression may be performed on mice deficient for Notchl or Notch2 in specific tissues through the use of conditional Notch alleles. Since alterations in specific Notch proteins are implicated in a variety of cancers and developmental syndromes, deciphering the regulation of Notch specific functions will have important implications for human health as Notch-specific therapy will have less untoward effects than global inhibition of Notch signaling. Relevance: Notch signaling plays an important role in development, determining the type of cells generated from embryonic stem cells, and alterations in its function have been linked to various forms of cancer. A more thorough understanding of Notch function will aid in the development of stem cell therapy, the generation of drugs to treat developmental disorders and cancers caused by malfunctioning Notch signaling, and avoid side-effects caused by inhibition of Notch signaling of potential therapies for Alzheimer's disease.